Intracellular retention of the T-cell receptor β chain in CD4⁺CD8⁺ double-positive and CD8 single-positive thymocytes of tamoxifen-treated lck-ER^T2-SCL transgenic mice. (A) After cell surface staining for CD4, CD8, and TCRβ (anti-TCRβ-FITC, TCRβ surf) thymocytes of tamoxifen (TAM)–treated (7 weeks) lck-ER^T2-SCL (n = 3) and wild-type control littermates (n = 3) were permeabilized and subjected to a second staining with anti-TCRβ-PE (TCRβ surf + ic). Anti-TCRβ-PE staining (TCRβ surf + ic) of the permeabilized thymocytes predominantly reflects intracellular staining because surface TCRβ epitopes were already saturated with the TCRβ-FITC antibody (same clone as TCRβ-PE) during the surface staining procedure. Levels of intracellular TCRβ expression (TCRβ surf + ic) were determined within surface DP (double-positive; CD4⁺CD8⁺) TCRβ^low, DP TCRβ^high, CD8⁺TCRβ^low, and CD8⁺TCRβ^high populations. Representative plots are shown. Dashed lines represent corresponding isotype control data. (B) Quantification (median fluorescent intensity, MFI) of TCRβ surface (surf) and TCRβ intracellular (surf + ic) expression. Bars represent means plus or minus SD; *P < .05; **P < .001. (C) Surface DP TCRβ^high CD8⁺TCRβ^low cells were analyzed for the presence of intracellular (surf + ic) variable β chains 2, 4, and 14 (Vβ2, Vβ4, and Vβ14). TAM-treated (13 weeks) lck-ER^T2-SCL (n = 3) and wild-type control littermates (n = 4) were analyzed. Representative plots are shown.